Estimation of polyacrylamide gel pore size from Ferguson plots of normal and anomalously migrating DNA fragments. I. Gels containing 3% N,N'-methylenebisacrylamide

The mobilities of normal and anomalously migrating DNA fragments were determined in polyacrylamide gels of different acrylamide concentrations, polymerized with 3% N,N'-methylenebisacrylamide as the crosslinker. The DNA samples were a commercially available 123-bp ladder and two molecular weight ladders containing multiple copies of two 147-base pair (bp) restriction fragments, obtained from the MspI digestion of plasmid pBR322. One of the 147 bp fragments is known to migrate anomalously slowly in polyacrylamide gels. Ferguson plots were constructed for all multimer ladders, using both absolute mobilities and relative mobilities with respect to the smallest DNA molecule in each data set. If the retardation coefficients were calculated from the relative mobilities, and the rms radius of gyration was used as the measure of DNA size, the Ogston equations were obeyed and the gel fiber parameters could be calculated. The effective pore sizes of the gels were estimated from the gel concentration at which the mobility of a given DNA molecule was reduced to one-half its mobility at zero gel concentration. The estimated pore radii ranged from approximately 130 nm for 3.5% gels to approximately 70 nm for 10.5% gels. These values are much larger than the pore sizes previously determined for the polyacrylamide matrix.     

Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

The electrophoretic mobilities of curved and normal DNA molecules of the same size have been measured in polyacrylamide gels containing various acrylamide concentrations and cross-linker ratios. Ferguson plots were constructed to extrapolate the observed mobilities to zero gel concentration. The DNA samples were two 147-bp restriction fragments, called 12A and 12B, obtained from the MspI digestion of plasmid pBR322, and head-to-tail multimers of each fragment. Fragment 12A is stably curved and migrates anomalously slowly in polyacrylamide gels; fragment 12B has the conformation of normal DNA and migrates with normal electrophoretic mobilities. The extrapolated mobilities of the curved fragment 12A and its multimers at zero gel concentration are lower than the extrapolated mobilities of the normal fragment 12B and its multimers. The free solution mobility of the curved fragment 12A, measured by CE, is also lower than that of the normal fragment 12B. The combined results indicate that the extrapolated mobilities observed for curved DNA molecules at zero polyacrylamide gel concentration reflect the intrinsic differences in their free solution mobilities.     

The electric field dependence of DNA mobilities in agarose gels: a reinvestigation

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments less than or equal to 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained less than or equal to 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incorporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments less than or equal to 12 kbp in size in agarose gels less than or equal to 1.4% in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 1 kbp in size in agarose gels greater than or equal to 2% in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an "intrinsic" DNA mobility of 2.7 x 10(-4) cm2/Vs at 20 degrees C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ferguson plots based on absolute mobilities in polyacrylamide gel electrophoresis: dependence of linearity of polymerization conditions and application to the determination of free mobility

In contrast to Ferguson plots based on relative mobilities, Ferguson plots of proteins in polyacrylamide gel electrophoresis based on their absolute mobilities were found to be linear under unusual polymerization conditions which yield relatively wide gel fibers and a low total fiber length per unit weight, but not under previously and commonly used conditions. These linear Ferguson plots in gels of 1, 3 and 5% crosslinking intersect at a single gel concentration between 1 and 2% T (M-point). It is postulated that the measure of free mobility of the proteins is the M-point, and not the intercept of their Ferguson plots with the mobility axis as assumed previously. This postulate abolishes the well-known paradoxical interpretation of the increase with %C of the linearly extrapolated intercept of the Ferguson plot with the log(mobility) axis (designated Yo) in terms of free mobility. The postulate is also compatible with the interpretation of the points of intersection of the Ferguson plots of oligomeric series of proteins at finite gel concentrations (designated mu-points) as their common free mobilities.     

Free mobility determination by electrophoresis in polyacrylamide containing agarose at a nonrestrictive concentration

In the determination of the free mobility, related to the surface net charge, by quantitative gel electrophoresis, the previous arbitrary extrapolation of Ferguson plots from the lowest gel concentrations that give a mechanically stable gel to 0% T has recently been replaced by measurement of mobilities across that concentration range, using the addition of 0.5% agarose to polyacrylamide at the various low concentrations in application to a DNA fragment 155 bp in size (Orbán, L. et al., in preparation). The present study applies that approach to several proteins and DNA fragments smaller than 1300 bp, using 0.4% agarose in polyacrylamide gels of varying concentration. The intercepts of the plots with the mobility axis provide experimental data by which the free mobility in polyacrylamide gel electrophoresis can be estimated for molecules not significantly retarded in their migration at the agarose concentration admixed to polyacrylamide. Across the gel concentration range below 3% T, in the presence of agarose, the Ferguson plots of proteins and DNA fragments are convex. It was shown by mass spectrometry that this convex curvature of the plots in the mixed polymer is not significantly due to low polymerization efficiency in the concentration range of liquid polyacrylamide (below 3%T).     

Transverse pore gradient gel electrophoresis of lipoprotein [a] using methylenebisacrylamide gradients.

We describe a modification of transverse pore gradient gel electrophoresis in which pore size is regulated by crosslinker proportion (%C) rather than by total monomer concentration (%T). We electrophoresed plasma lipoprotein [a] transversely across linear N,N'-methylenebisacrylamide gradients and measured mobility as a function of %C. This method allows for the simultaneous assessment of pore sizes generated over a wide range of crosslinker proportions.     

Effect of total percent polyacrylamide in capillary gel electrophoresis for DNA sequencing of short fragments. A phenomenological model.

Polyacrylamide capillary gels were prepared with constant (5% C) cross-linker concentration and with total acrylamide concentration ranging from 2.5 to 6% T. At each acrylamide concentration, peak spacing was constant for DNA sequencing fragments ranging from 25 to 250 nucleotides in length. Peak spacing increased linearly with the total acrylamide concentration. The intercept of the retention time vs. fragment length plot was independent of % T. Ferguson plots were constructed for short DNA fragments; the polyacrylamide pore size falls in the 2.5 to 3.5 nm range for the gels studied. Theoretical plate count is independent of total acrylamide concentration; longitudinal diffusion, and not thermal gradients, limit the plate count. A phenomenological model is presented that predicts retention time, plate count, and resolution for sequencing fragments ranging in size from 25 to 250 bases and gels that range from 2.5 to 6% total acrylamide.     

Resolution of 8-aminonaphthalene-1,3,6-trisulfonic acid-labeled glucose oligomers in polyacrylamide gel electrophoresis at low gel concentration

A discontinuous Tris-Cl/acetate (OAc) buffer system, unprecedently containing OAc as the trailing constituent, and operative in polyacrylamide gel electrophoresis (PAGE) at low polyacrylamide concentration (T = 4.8%) is described in the paper. The characteristics of the electrophoretic system are illustrated by the resolution of fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled malto-oligosaccharides and dextran homopolymers. In this buffer system, the resolving phase is constituted by Tris-OAc behind a moving boundary formed between the leading chloride ion of Tris-HCl gel buffer and the trailing OAc ion provided by a catholyte of NH(4)OAc. In contrast with the results obtained with Tris-CI/glycinate buffer commonly used in electrophoresis, or with Tris-CI/borate, the best resolution of the glucose oligomers containing 1-4 glucose units in Tris-OAc, pH 8.8, ionic strength of 0.08, was obtained at 4.8% polyacrylamide concentration, using 0.5 M NH(4)OAc, pH 9.5 as the catholyte. Under those conditions, the ANTS-glucose oligomers were separated with mobilities decreasing from glucose to maltohexaose. The linear Ferguson plots (log relative mobility, R(f), vs.%T) of the glucose oligomers show that the surface net charge of those oligomers is inversely related to their sizes, given by the slopes, K(R), of the plots. The molecular weight of the oligomers is directly but nonlinearly related to K(R). The novel electrophoretic system illustrated here for separation of short ANTS-saccharides can be potentially applied to the resolution of other biomolecules such as rapidly migrating DNA, peptides or proteins.     

Novel cross-linked polyacrylamide matrices: an investigation using gradient gel electrophoresis

Gradient gel electrophoresis was used to examine the separation properties of novel cross-linking compounds for polyacrylamide (PAAm). At low %T and at the same %C protein migration difference is accentuated for bismethacrylamide cross-linked networks relative to bisacrylamide cross-linked networks. Similar properties were observed for cyclic monomers at low %T. This trend is maintained throughout the gradient. However, at higher %T migration differential relative to N,N'-methylenebisacrylamide (Bis) was less pronounced. Evidence from gradient gels suggests that reactivity and functionality of vinyl groups impose an overriding control over network formation.     

Sieving of ionic constituents across moving boundaries in gel electrophoresis

The representative beta-hydroxyethylmorpholinium-chloride-bicinate moving boundary with a trailing ion net mobility relative to Na+ of 0.41, detected by precipitation of chloride with silver nitrate, exhibits a decreasing chloride mobility at increasing polyacrylamide gel concentrations from 3.5 to 45%T, 5%CBis. This decrease, largely due to an increase of field strength at constant current, is described by a convex* plot of log (mobility) vs. %T (Ferguson plot) and signifies that chloride/bicinate are sieved by the gel. In agarose gels, the same plot of mobility vs. gel concentration is constant below 7% gel concentration, since in those gels field strength and migration rate remain the same within that gel concentration range. Both in polyacrylamide and in agarose gels the displacement rate of the chloride-bicinate boundary as a function of the time of electrophoresis or distance migrated remains invariant within 15%. The plot of log (mobility) vs. gel concentration extrapolated to 0%T is 5.85 and 5.41 (10(-5) cm2s-1V-1) for polyacrylamide and for agarose (SeaKem HGT-P,FMC) gels, respectively. The slightly decreased mobility intercept at 0%T for agarose is presumably due either to the electroendosmotic properties of agarose HGT-P and/or failure to Sufficiently take into account the flattening of the Ferguson plot in the polyacrylamide concentration range below 3% in which a transition from a gel to a fluid (sol) medium takes place.     

'Laterally aggregated' polyacrylamide gels for electrophoresis.

A new method is described for producing highly porous polyacrylamide matrices: polymerization in presence of a preformed hydrophilic polymer. If a standard mixture of monomers (e.g., 5%T, 4%C) is polymerized in presence of, e.g., polyethylene glycol (PEG) 10 kDa, lateral chain aggregation occurs, with formation of large pore sizes. In PEG 10 kDa, the transition from a small- to a large-pore gel is clearly apparent at 0.5% PEG addition and reaches a plateau already at 2.5% PEG. Even with shorter PEG fragments (6.2 and 1 kDa) this transition occurs, but with progressively larger amounts of PEG in solution (up to 25% for the 1 kDa species). Other polymers such as hydroxymethyl cellulose (1000 kDa) and polyvinyl-pyrrolidone (360 kDa and 25 kDa) are also able to elicit this phenomenon. It appears that lateral chain aggregation (before the cross-linking event) is induced via intra-chain hydrogen bonding, since urea and temperature strongly inhibit it, whereas tetramethylurea (an agent quenching hydrophobic interactions) does not hamper it. By scanning electron microscope, it is found that the maximum pore size obtained in a 5%T, 4%C gel in presence of 2.5% PEG 10 kDA is of the order of 0.5 micron, whereas the same 5%T, 4%C control gel would have an average pore diameter of 5 nm. Thus, an increment of pore size of about 2 orders of magnitude is obtained: in these new matrices, a 21000 bp DNA fragment exhibits a much greater migration than in a control gel in which the sample is entrapped at the application site.     

Information on DNA conformation derived from transverse pore gradient gel electrophoresis in conjunction with an advanced data analysis applied to capillary electrophoresis in polymer media.

Abnormally slow migration of DNA is conventionally viewed as being due to an abnormal conformation relative to "linear" standards. The evidence for this rests on a few instances where nonlinear DNA structures have been established by independent methods and yield low mobilities relative to standards. Transverse pore gradient gel electrophoresis of authentically bent kinetoplast DNA and of an upstream activator sequence (UAS) of an E. coli operon promoter shows in addition that curves of migration distance vs. gel concentration ("Ferguson curves") of such abnormally conformed DNA differ from those of "linear" standards. Since Ferguson curves are interpretable with regard to molecular size in concordance with a mathematical model (Ogston model), transverse pore gradient gel electrophoresis provides a simple means of correlating abnormally slow migration of DNA with molecular size. In addition, transverse pore gradient gel electrophoresis is able to distinguish between DNA banding which exhibits a steeper dependence on gel concentration than "linear" standards from one which shows the same dependence. The former appears characteristic of circularly bent DNA and gives rise to a substantial retardation, the latter of bending across a knot or kink in the DNA chain associated with a relatively minor retardation relative to standards. Circularly bent restriction fragments formed from kinetoplast DNA retain the characteristic intersecting Ferguson curves on the transverse pore gradient gel. Another authentically "abnormal" DNA structure recognizable on transverse pore gradient gels is supercoiled DNA derived from the reaction of topoisomerase with a plasmid. Different lengths of supercoiled sequences give rise to parallel Ferguson curves clearly intersecting with those of linear standards.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields

This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacrylamide with low amounts of crosslinking agent (i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels (e.g. 5% C), permitted high efficiency separations of double stranded DNA fragments up to 12,000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots (i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacrylamide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation.     

Molecular sieving of lambda phage DNA in polyacrylamide solutions as a function of the molecular weight of the polymer.

Electrophoresis of lambda phage DNA was carried out in solutions at various concentrations of uncrosslinked polyacrylamide of 0.6, 1, 5 and 9 x 10(6) molecular weight (Mw) with narrow Mw distribution. By inspection of mobilities in the various concentration ranges, it appears that mobilities decrease, and retardation increases, with increasing Mw. The relation between electrophoretic retardation and the Mw of the polymer was also interpreted (i) in the manner previously applied to nonlinear Ferguson plots and compatible with the Ogston model; and (ii) empirically, on the basis of the first derivatives of the functions describing the Ferguson plots at the polymer concentrations used. Interpretation (i) shows that the retardation increases linearly in the order of 0.6, 1, 5 and 9 x 10(6) Mw of polyacrylamide. Interpretation (ii) shows a nonlinear increase of retardation in the Mw range 5 to 9 x 10(6), and a decrease in retardation as Mw is raised from 0.6 to 5.0 x 10(6). Hypothetically, interpretation (ii) can be explained mechanistically by a progressive change, as the polymer size is increased, from a collision with the surface of the polymer fiber to one occurring after permeation in the interior of a random-coiled fiber. Interpretation (i) may fail to detect that change due to the large difference between DNA mobility in solutions of the smallest polymer and the free mobility. DNA peak detection in all of the four size classes of polyacrylamide in solution is limited to relatively narrow ranges of polymer concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel polyacrylamide gel system for proteomic use offering controllable pore expansion by crosslinker cleavage

SDS‐PAGE is still one of the most widespread separation techniques in proteomic research and usually coupled to subsequent MS measurement for protein identification. The proteins are digested while embedded in the gel matrix. The resultant peptides are eluted out of the gel and finally analyzed. The in‐gel digestion process suffers from several drawbacks which influence the experimental outcome with respect to protein sequence coverage and detection sensitivity. Limited accessibility of the protease to the substrate protein and insufficient peptide extraction represent the two major problems. To specifically target these issues, we established a novel partly reversible gel system, in which the gel matrix can be conditionally cleaved to increase the pore diameters. By using a crosslinker mixture consisting of Bis and ethylene‐glycol‐diacrylate the acrylamide filament interconnections can be partly hydrolyzed in alkaline solution. The new hybrid gels have been tested to be compatible with a variety of acidic staining techniques. They exhibit similar electrophoretic performance compared with regular solely Bis‐based gels, but yield significantly better MS results. Thus, the Bis/ethylene‐glycol‐diacrylate SDS‐PAGE gel system is a promising alternative for MS‐based in‐gel workflows and might be transferred to other gel‐electrophoretic applications.     

Resolution of a paradox in the electrophoresis of DNA in agarose gels

A paradox was observed in a previous study of the electrophoresis of linear DNA fragments in agarose gels (D. L. Holmes and N. C. Stellwagen, Electrophoresis 1990, 11, 5-15). The pore size of the agarose matrix was more accurately determined if the root-mean-square radius of gyration was used to measure DNA macromolecular size. However, the Ogston equations were obeyed and other gel parameters such as the apparent fiber radius and fiber volume appeared to be better described if the geometric mean radius was used to measure DNA size. This paradox can be resolved if relative mobilities (with respect to the smallest DNA molecule in the data set) are used to construct the Ferguson plots, instead of absolute mobilities. Using relative mobilities and the root-mean-square radius of gyration, the Ogston equations are obeyed and the pore size of the matrix is consistent with values determined by other methods.     

Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide.

Proteins with relative molecular masses of 14,000 to 205,000 were separated by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using non-cross-linked linear polyacrylamide gels on both coated and uncoated fused-silica capillaries. It was determined that viscosity of the acrylamide solution was a major factor affecting column stability with linear acrylamide gels. When the viscosity of the acrylamide solution reaches 100 cP, electro-osmotically driven displacement of the gels is insignificant. Uncoated capillaries provided better resolution, stability, and reproducibility than surface coated capillaries when the concentration of linear polyacrylamide was greater than 4%. At lower gel concentrations, non-cross-linked polyacrylamide is easily displaced from the columns. A calibration plot of log molecular mass vs. mobility with non-linear polyacrylamide was linear, which indicated that resolution was equivalent to that obtained with cross-linked acrylamide. Separations with model proteins indicated that baseline resolution between protein species that vary 10% in molecular mass can be achieved.     

Nanotube-grafted polyacrylamide hydrogels for electrophoretic protein separation

Multiwalled carbon nanotube-modified polyacrylamide gels have been employed for the electrophoretic separation of proteins. Two approaches are compared in this investigation, one using nanotubes only as fillers inside the gel matrix and the other using nanotubes as catalyst for polymerization of acrylamide. In both the cases, polymerization of acryl-amide/bisacrylamide has been carried out in situ in the presence of nanotubes dispersed in the gel buffer containing monomer and cross-linker. In the former case, initiator and catalyst have been added after ultrasonication of nanotubes in the gel buffer mixture where the nanotubes play the role of filler. On the other hand, the second approach precludes use of catalyst and involves addition of initiator alone during ultrasonication of nanotubes in the gel buffer containing monomer and cross-linker, which leads to the formation of nanotube-grafted gel after 25 min. When nanotubes are used as a catalyst instead of N,N,N',N'-tetramethylethylenediamine, pore size distribution of the gel matrix and linearity of molecular weight calibration plots are found to be improved. In addition, other issues associated with the use of an external catalyst like handling the moisture-sensitive and corrosive reagent and associated irreproducibility are addressed in this approach.     

Procedures and computer program for deriving the Ferguson plot from electrophoresis in a single pore gradient gel: application to agarose gel and a polystyrene particle.

This study presents a computerized evaluation of pore gradient gel electrophoretograms to arrive at estimates for both the particle-free mobility and retardation coefficient, which is related to particle size. Agarose pore gradient gels ranging from 0.2 to 1.1% agarose were formed. Gel gradients were stabilized during their formation by a density gradient of 0-20% 5-(N-2,3-dihydroxypropylacetamido)- 2,4,6-triiodo-N,N'bis-(2,3-dihydroxypropyl)-isophthalamide (Nycodenz). Densitometry of gelled-in Bromophenol Blue showed that these pore gradients exhibited a linear central segment and were reproducible. Migration distances of polystyrene sulfate microspheres (36.5 nm radius) in agarose pore gradient gel electrophoresis were determined by time-lapse photography at several durations of electrophoresis. These migration distances were evaluated as a function of migration time as previously reported (D. Tietz, Adv. Electrophoresis 1988, 2, 109-169). Although this is not necessarily required, the mathematical approach used in this study assumed linearity of both the pore gradient and the Ferguson plot for reasons of simplicity. The data evaluation on the basis of the extended Ogston model is incorporated in a user-friendly program, GRADFIT, which is designed for personal computers (Macintosh). The results obtained are compared with (1) conventional electrophoresis using several gels of single concentration with and without Nycodenz, and (ii) a different mathematical approach for the analysis of gradient gels (Rodbard et al., Anal. Biochem. 1971, 40, 135-157). Moreover, a simple procedure for evaluating linear pore gradient gels using linear regression analysis is presented. It is concluded that the values of particle-free mobility and retardation coefficient derived from pore gradient gel electrophoresis using the different mathematical methods are statistically indistinguishable from each other. However, these values are different, albeit close, to those obtained from conventional Ferguson plots. One of the possible reasons for this relatively minor discrepancy is that the particle-free mobility changed slightly during electrophoresis, which has a different effect on electrophoresis in homogeneous gels (single time measurement) and pore gradient gels (multiple time measurements). The characterization of particles according to size and charge by pore gradient electrophoresis provides a significant operational simplification and sample economy compared to that requiring the use of several gel concentrations, although at the price of increased requirements of instrumentation.     

Concave Ferguson plots of DNA fragments and convex Ferguson plots of bacteriophages: evaluation of molecular and fiber properties, using desktop computers.

A desktop computer program evaluating physical properties of DNA and bacteriophages is presented. The analysis is based on data obtained from capillary and submarine-type agarose electrophoresis. Native molecular/particle properties and properties of the gel (or polymer) medium can be derived from electrophoresis at several gel concentrations. This is done conveniently by a computerized evaluation of the semi-logarithmic plot of mobility vs. gel concentration, designated the Ferguson plot. In application to most proteins, this plot is linear and computer programs exist to evaluate it. However, nonlinear Ferguson plots have assumed great importance in view of the fact that the plots are concave for DNA. Similarly, convex plots are important since they prevail in the electrophoresis of large particles in agarose. The computer program reported here is the first to (i) address concave Ferguson plots and (ii) allow for the evaluation of both cases using a desktop computer. Program ELPHOFIT version 2.0, a Macintosh application, is available upon request.